Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19.

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.

The Stability of an Isolate of the SARS-CoV-2 B.1.1.7 Lineage in Aerosols Is Similar to 3 Earlier Isolates.

BACKGROUND: Our laboratory previously examined the influence of environmental conditions on the stability of an early isolate of SARS-CoV-2 (hCoV-19/USA/WA-1/2020) in aerosols generated from culture medium or simulated saliva. However, genetic differences have emerged among SARS-CoV-2 lineages, and it is possible that these differences may affect environmental stability and the potential for aerosol transmission. METHODS: The influence of temperature, relative humidity, and simulated sunlight on the decay of 4 SARS-CoV-2 isolates in aerosols, including 1 belonging to the recently emerged B.1.1.7 lineage, were compared in a rotating drum chamber. Aerosols were generated from simulated respiratory tract lining fluid to represent aerosols originating from the deep lung. RESULTS: No differences in the stability of the isolates were observed in the absence of simulated sunlight at either 20°C or 40°C. However, a small but statistically significant difference in the stability was observed between some isolates in simulated sunlight at 20°C and 20% relative humidity. CONCLUSIONS: The stability of SARS-CoV-2 in aerosols does not vary greatly among currently circulating lineages, including B.1.1.7, suggesting that the increased transmissibility associated with recent SARS-CoV-2 lineages is not due to enhanced survival in the environment.

Simulated Sunlight Rapidly Inactivates SARS-CoV-2 on Surfaces.

Previous studies have demonstrated that SARS-CoV-2 is stable on surfaces for extended periods under indoor conditions. In the present study, simulated sunlight rapidly inactivated SARS-CoV-2 suspended in either simulated saliva or culture media and dried on stainless steel coupons. Ninety percent of infectious virus was inactivated every 6.8 minutes in simulated saliva and every 14.3 minutes in culture media when exposed to simulated sunlight representative of the summer solstice at 40°N latitude at sea level on a clear day. Significant inactivation also occurred, albeit at a slower rate, under lower simulated sunlight levels. The present study provides the first evidence that sunlight may rapidly inactivate SARS-CoV-2 on surfaces, suggesting that persistence, and subsequently exposure risk, may vary significantly between indoor and outdoor environments. Additionally, these data indicate that natural sunlight may be effective as a disinfectant for contaminated nonporous materials.

Airborne SARS-CoV-2 Is Rapidly Inactivated by Simulated Sunlight.

Aerosols represent a potential transmission route of COVID-19. This study examined effect of simulated sunlight, relative humidity, and suspension matrix on stability of SARS-CoV-2 in aerosols. Simulated sunlight and matrix significantly affected decay rate of the virus. Relative humidity alone did not affect the decay rate; however, minor interactions between relative humidity and other factors were observed. Mean decay rates (± SD) in simulated saliva, under simulated sunlight levels representative of late winter/early fall and summer were 0.121 ±â€…0.017 min-1 (90% loss, 19 minutes) and 0.306 ±â€…0.097 min-1 (90% loss, 8 minutes), respectively. Mean decay rate without simulated sunlight across all relative humidity levels was 0.008 ±â€…0.011 min-1 (90% loss, 286 minutes). These results suggest that the potential for aerosol transmission of SARS-CoV-2 may be dependent on environmental conditions, particularly sunlight. These data may be useful to inform mitigation strategies to minimize the potential for aerosol transmission.

Exposure of rhesus monkeys to cowpox virus Brighton Red by large-particle aerosol droplets results in an upper respiratory tract disease.

We previously demonstrated that small-particle (0.5-3.0 µm) aerosol infection of rhesus monkeys (Macaca mulatta) with cowpox virus (CPXV)-Brighton Red (BR) results in fulminant respiratory tract disease characterized by severe lung parenchymal pathology but only limited systemic virus dissemination and limited classic epidermal pox-like lesion development (Johnson et al., 2015). Based on these results, and to further develop CPXV as an improved model of human smallpox, we evaluated a novel large-particle aerosol (7.0-9.0 µm) exposure of rhesus monkeys to CPXV-BR and monitored for respiratory tract disease by serial computed tomography (CT). As expected, the upper respiratory tract and large airways were the major sites of virus-induced pathology following large-particle aerosol exposure. Large-particle aerosol CPXV exposure of rhesus macaques resulted in severe upper airway and large airway pathology with limited systemic dissemination.

Aerosol Particle Size Influences the Infectious Dose and Disease Severity in a Golden Syrian Hamster Model of Inhalational COVID-19.

Background: Significant evidence suggests that SARS-CoV-2 can be transmitted via respiratory aerosols, which are known to vary as a function of respiratory activity. Most animal models examine disease presentation following inhalation of small-particle aerosols similar to those generated during quiet breathing or speaking. However, despite evidence that particle size can influence dose-infectivity relationships and disease presentation for other microorganisms, no studies have examined the infectivity of SARS-CoV-2 contained in larger particle aerosols similar to those produced during coughing, singing, or talking. Therefore, the aim of the present study was to assess the influence of aerodynamic diameter on the infectivity and virulence of aerosols containing SARS-CoV-2 in a hamster model of inhalational COVID-19. Methods: Dose-response relationships were assessed for two different aerosol particle size distributions, with mass median aerodynamic diameters (MMADs) of 1.3 and 5.2 µm in groups of Syrian hamsters exposed to aerosols containing SARS-CoV-2. Results: Disease was characterized by viral shedding in oropharyngeal swabs, increased respiratory rate, decreased activity, and decreased weight gain. Aerosol particle size significantly influenced the median doses to induce seroconversion and viral shedding, with both increasing ∼30-fold when the MMAD was increased. In addition, disease presentation was dose-dependent, with seroconversion and viral shedding occurring at lower doses than symptomatic disease characterized by increased respiratory rate and decreased activity. Conclusions: These results suggest that aerosol particle size may be an important factor influencing the risk of COVID-19 transmission and needs to be considered when developing animal models of disease. This result agrees with numerous previous studies with other microorganisms and animal species, suggesting that it would be generally translatable across different species. However, it should be noted that the absolute magnitude of the observed shifts in the median doses obtained with the specific particle sizes utilized herein may not be directly applicable to other species.

SARS-CoV-2 inactivation by ultraviolet radiation and visible light is dependent on wavelength and sample matrix.

Numerous studies have demonstrated that SARS-CoV-2 can be inactivated by ultraviolet (UV) radiation. However, there are few data available on the relative efficacy of different wavelengths of UV radiation and visible light, which complicates assessments of UV decontamination interventions. The present study evaluated the effects of monochromatic radiation at 16 wavelengths from 222 nm through 488 nm on SARS-CoV-2 in liquid aliquots and dried droplets of water and simulated saliva. The data were used to generate a set of action spectra which quantify the susceptibility of SARS-CoV-2 to genome damage and inactivation across the tested wavelengths. UVC wavelengths (≤280 nm) were most effective for inactivating SARS-CoV-2, although inactivation rates were dependent on sample type. Results from this study suggest that UV radiation can effectively inactivate SARS-CoV-2 in liquids and dried droplets, and provide a foundation for understanding the factors which affect the efficacy of different wavelengths in real-world settings.

Comparison of Dose-Response Relationships for Two Isolates of SARS-CoV-2 in a Nonhuman Primate Model of Inhalational COVID-19.

Background: As the COVID-19 pandemic has progressed, numerous variants of SARS-CoV-2 have arisen, with several displaying increased transmissibility. Methods: The present study compared dose-response relationships and disease presentation in nonhuman primates infected with aerosols containing an isolate of the Gamma variant of SARS-CoV-2 to the results of our previous study with the earlier WA-1 isolate of SARS-CoV-2. Results: Disease in Gamma-infected animals was mild, characterized by dose-dependent fever and oronasal shedding of virus. Differences were observed in shedding in the upper respiratory tract between Gamma- and WA-1-infected animals that have the potential to influence disease transmission. Specifically, the estimated median doses for shedding of viral RNA or infectious virus in nasal swabs were approximately 10-fold lower for the Gamma variant than the WA-1 isolate. Given that the median doses for fever were similar, this suggests that there is a greater difference between the median doses for viral shedding and fever for Gamma than for WA-1 and potentially an increased range of doses for Gamma over which asymptomatic shedding and disease transmission are possible. Conclusions: These results complement those of previous studies, which suggested that differences in exposure dose may help to explain the range of clinical disease presentations observed in individuals with COVID-19, highlighting the importance of public health measures designed to limit exposure dose, such as masking and social distancing. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, as well as to inform dose selection in future studies examining the efficacy of therapeutics and vaccines in animal models of inhalational COVID-19.

The influence of temperature, humidity, and simulated sunlight on the infectivity of SARS-CoV-2 in aerosols.

Recent evidence suggests that respiratory aerosols may play a role in the spread of SARS-CoV-2 during the ongoing COVID-19 pandemic. Our laboratory has previously demonstrated that simulated sunlight inactivated SARS-CoV-2 in aerosols and on surfaces. In the present study, we extend these findings to include the persistence of SARS-CoV-2 in aerosols across a range of temperature, humidity, and simulated sunlight levels using an environmentally controlled rotating drum aerosol chamber. The results demonstrate that temperature, simulated sunlight, and humidity are all significant factors influencing the persistence of infectious SARS-CoV-2 in aerosols, but that simulated sunlight and temperature have a greater influence on decay than humidity across the range of conditions tested. The time needed for a 90% decrease in infectious virus ranged from 4.8 min at 40 °C, 20% relative humidity, and high intensity simulated sunlight representative of noon on a clear day on the summer solstice at 4°N latitude, to greater than two hours under conditions representative of those expected indoors or at night. These results suggest that the persistence of infectious SARS-CoV-2 in naturally occurring aerosols may be affected by environmental conditions, and that aerosolized virus could remain infectious for extended periods of time under some environmental conditions. The present study provides a comprehensive dataset on the influence of environmental parameters on the survival of SARS-CoV-2 in aerosols that can be utilized, along with data on viral shedding from infected individuals and the inhalational infectious dose, to inform future modeling and risk assessment efforts.

Aerosol exposure to intermediate size Nipah virus particles induces neurological disease in African green monkeys.

Nipah virus (NiV) infection can lead to severe respiratory or neurological disease in humans. Transmission of NiV has been shown to occur through contact with virus contaminated fomites or consumption of contaminated food. Previous results using the African green monkey (AGM) model of NiV infection identified aspects of infection that, while similar to humans, don't fully recapitulate disease. Previous studies also demonstrate near uniform lethality that is not consistent with human NiV infection. In these studies, aerosol exposure using an intermediate particle size (7µm) was used to mimic potential human exposure by facilitating virus deposition in the upper respiratory tract. Computed tomography evaluation found some animals developed pulmonary parenchymal disease including consolidations, ground-glass opacities, and reactive adenopathy. Despite the lack of neurological signs, magnetic resonance imaging identified distinct brain lesions in three animals, similar to those previously reported in NiV-infected patients. Immunological characterization of tissues collected at necropsy suggested a local pulmonary inflammatory response with increased levels of macrophages in the lung, but a limited neurologic response. These data provide the first clear evidence of neurological involvement in the AGM that recapitulates human disease. With the development of a disease model that is more representative of human disease, these data suggest that NiV infection in the AGM may be appropriate for evaluating therapeutic countermeasures directed at virus-induced neuropathogenesis.

Influenza A and methicillin-resistant Staphylococcus aureus co-infection in rhesus macaques - A model of severe pneumonia.

BACKGROUND: Influenza results in up to 500,000 deaths annually. Seasonal influenza vaccines have an estimated 60% effectiveness, but provide little or no protection against novel subtypes, and may be less protective in high-risk groups. Neuraminidase inhibitors are recommended for the treatment of severe influenza infection, but are not proven to reduce mortality in severe disease. Preclinical models of severe influenza infection that closely correlate to human disease are needed to assess efficacy of new vaccines and therapeutics. METHODS: We developed a nonhuman primate model of influenza and bacterial co-infection that recapitulates severe pneumonia in humans. Animals were infected with influenza A virus via intra-bronchial or small-particle aerosol inoculation, methicillin-resistant Staphylococcus aureus, or co-infected with influenza and methicillin-resistant S. aureus combined. We assessed the severity of disease in animals over the course of our study using tools available to evaluate critically ill human patients including high-resolution computed tomography imaging of the lungs, arterial blood gas analyses, and bronchoalveolar lavage. RESULTS: Using an intra-bronchial route of inoculation we successfully induced severe pneumonia following influenza infection alone and following influenza and bacterial co-infection. Peak illness was observed at day 6 post-influenza infection, manifested by bilateral pulmonary infiltrates and hypoxemia. The timing of radiographic and physiologic manifestations of disease in our model closely match those observed in severe human influenza infection. DISCUSSION: This was the first nonhuman primate study of influenza and bacterial co-infection where high-resolution computed tomography scanning of the lungs was used to quantitatively assess pneumonia over the course of illness and where hypoxemia was correlated with pneumonia severity. With additional validation this model may serve as a pathway for regulatory approval of vaccines and therapeutics for the prevention and treatment of severe influenza pneumonia.

Small particle aerosol inoculation of cowpox Brighton Red in rhesus monkeys results in a severe respiratory disease.

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases.